The mammalian transcription factors Sp1 and Sp3 compete for the same DNA binding sites but play different roles in the regulation of expression of numerous genes. It is known that, in the interphase nucleus, Sp1 and Sp3 are organized into distinct foci. In this study, we show that throughout the mitotic process, while being displaced from the condensed chromosomes and dispersed throughout the cell, Sp1 and Sp3 maintain their separate punctate distributions. In metaphase, both Sp1 and Sp3 foci show a high degree of colocalization with microfilaments, suggesting that F-actin is involved in the organization of Sp1 and Sp3 foci during mitosis. Constant Sp1 and Sp3 levels were observed during mitosis, signifying a recovery of the pre-existing Sp1 and Sp3 population in newly formed nuclei. In late telophase, Sp1 and Sp3 are equally segregated between daughter cells, and their subnuclear organization as distinct foci is restored in a sequential fashion with Sp3 regrouping into the newly formed nuclei prior to Sp1. Both Sp1 and Sp3 return to the nuclei ahead of RNA polymerase II. Our results support a model in which entry of Sp1, Sp3 and RNA polymerase II into the newly formed nuclei is an ordered process.